It is estimated that between 20-25% of American adults (about 47 million) have metabolic syndrome, a complex condition associated with an increased risk of vascular disease. Metabolic syndrome is also known as Syndrome X, metabolic syndrome X, insulin resistance syndrome, or Reaven's syndrome, after Dr. Gerald M. Reaven, who first described the disorder. Metabolic syndrome is generally believed to be a combination of disorders that affect a large number of people in a clustered fashion. The symptoms and features of the syndrome include at least three of the following conditions: diabetes mellitus type II; impaired glucose tolerance or insulin resistance; high blood pressure; central obesity and difficulty losing weight; high cholesterol; combined hyperlipidemia; including elevated LDL; decreased HDL; elevated triglycerides; and fatty liver (especially in concurrent obesity). Insulin resistance is typical of metabolic syndrome and leads to several of its features, including glucose intolerance, dyslipidemia, and hypertension. Obesity is commonly associated with the syndrome as is increased abdominal girth, highlighting the fact that abnormal lipid metabolism likely contributes to the underlying pathophysiology of metabolic syndrome.
Metabolic syndrome was codified in the United States with the publication of the National Cholesterol Education Program Adult Treatment Panel III (ATP III) guidelines in 2001. On a physiologic basis, insulin resistance appears to be responsible for the syndrome. However, insulin resistance can be defined in a myriad of different ways, including impaired glucose metabolism (reduced clearance of glucose and/or the failure to suppress glucose production), the inability to suppress lipolysis in tissues, defective protein synthesis, altered cell differentiation, aberrant nitric oxide synthesis affecting regional blood flow, as well as abnormal cell cycle control and proliferation, all of which have been implicated in the cardiovascular disease associated with metabolic syndrome. At least at present, there is no obvious molecular mechanism causing the syndrome, probably because the condition represents a failure of one or more of the many compensatory mechanisms that are activated in response to energy excess and the accumulation of fat.
According to ATP III, the diagnosis of metabolic syndrome requires the presence of three or more of the following: elevated fasting triglycerides (greater than or equal to 150 mg/dl), low HDL cholesterol (less than 50 mg/dl in women, less than 40 mg/dl in men), hypertension (blood pressure greater than or equal to 130/85 mm Hg), increased waist circumference (due to excess visceral adiposity, greater than 35 inches in women, greater than 40 inches in men) and elevated fasting glucose (greater than or equal to 100 mg/dl). The presence of three components is not a perfect predictor of insulin resistance, and the World Health Organization has established somewhat different criteria that include microalbuminuria (i.e., slightly elevated albumin excretion in the urine), and some groups modify the ATP III criteria to include a body mass index (BMI) of greater than or equal to 30 kg /M2 and abnormal nonfasting glucose and lipid values. Regardless of the definition, the syndrome identifies a group of individuals at increased risk for vascular disease. In an analysis of the Third National Health and Nutrition Examination Survey (NHANES III) participants over the age of 50 with metabolic syndrome showed a coronary heart disease prevalence exceeding that of diabetes. NHANES II data indicate total mortality as well as death from coronary heart disease and cardiovascular disease are increased in adults with metabolic syndrome.
Individuals at risk for metabolic syndrome include those who exhibit central obesity with increased abdominal girth (due to excess visceral adiposity) of about more than 35 inches in women and more than 40 inches in men. Individuals at risk for metabolic syndrome also include those that have a BMI greater than or equal to 30 kg/M2 and may also have abnormal levels of nonfasting glucose, lipids, and blood pressure.
Oxidative Stress, Metabolic Syndrome, and Vascular Disease
Reductionist systems, animal models and studies in humans are consistent with a role for reactive oxygen species in the development of vascular dysfunction. Chronic inflammation is one source of reactive oxygen species and an emerging body of evidence implicates inflammation and oxidative stress in vascular disease associated with the metabolic syndrome. CRP (C reactive protein), a circulating marker of inflammation, is higher in those with more components of the metabolic syndrome, and elevated CRP levels may be predictive of vascular disease events in women with the syndrome. Cytokines associated with insulin resistance, such as IL-6 and TNFα, also increase with obesity. Obesity promotes the accumulation of macrophages and an induction of an inflammatory pattern of gene expression in adipose tissue. Inhibition of oxidative stress pathways decreases reactive oxygen species in adipose tissue and improves metabolic syndrome in obese mouse models. Obesity increases expression of monocyte-chemoattractant protein-1, a proatherogenic molecule that also promotes insulin resistance.
JNK and the Metabolic Syndrome
While oxidative stress can affect multiple potential molecular mediators of the metabolic syndrome, c-Jun N-terminal kinase (JNK) may be particularly important. JNK, a member of the mitogen activated protein kinase superfamily of signaling molecules, is activated by stressors that include reactive oxygen species, fatty acids, and inflammatory cytokines. Activated JNK phosphorylates cJun, which in combination with cFos constitutes the AP-1 transcription factor complex. Several lines of evidence link JNK activity to features of the metabolic syndrome. JNK activity is increased by endoplasmic reticulum stress caused by obesity. There are three known JNK genes and multiple isoforms. JNK1 and 2 are widely distributed, while JNK3 expression is more limited. Mice deficient in JNK1 are protected from obesity and insulin resistance. Expression of wild type JNK in liver decreases insulin sensitivity and overexpression of a dominant negative JNK in liver increases insulin sensitivity in obese mice. JNK decreases the expression of adiponectin, which has insulin sensitizing effects, in adipocyte cell lines. Treatment of animals with a cell-permeable inhibitor of JNK enhances insulin sensitivity. At least part of the mechanism underlying the JNK effect is understood since JNK has been shown to interfere with insulin signaling by phosphorylating serine residue 307 on insulin receptor substrate 1 (IRS1). Pharmacologic inhibition of JNK activity and genetic JNK2 deficiency in apoE null mice decreases atherosclerosis, in part due to decreased activity of scavenger receptor A in the absence of JNK-dependent phosphorylation.
ATM and Stress Responses
Ataxia telangiectasia (AT) is an autosomal recessive disorder presenting in early childhood that is characterized by progressive cerebellar ataxia, skin and eye telangiectasias, a predisposition to malignancies (especially lymphomas), and immune deficiency. AT patients also manifest impaired growth, accelerated aging, and other signs of insulin resistance including glucose intolerance. The likely mechanism of AT was provided when the single gene responsible for this disease, ATM (Ataxia Telangiectasia Mutated) was identified and found to be a member of the phosphoinositol-3 kinase family. ATM was subsequently shown to be important for insulin signaling leading to translation initiation by Yang and Kastan in Nature Cell Bio., 2000, 2: 893-898. The frequency of ATM heterozygotes may be as high as 2% in the general population, and a study of these ATM carriers (405 grandparents of AT children) reported an increased risk of death from ischemic heart disease.
The primary function of ATM is to respond to DNA damage, much of which is caused by reactive oxygen species. After genotoxic stress, ATM initiates pathways that interfere with cell cycle progression, permitting DNA to be repaired before errors are propagated through replication. ATM is recognized to be important for restoring homeostasis in response to oxidative stress. Levels of manganese superoxide dismutase, catalase, and thioredoxin reflective of increased oxidative stress are present in cerebellae from ATM null mice. ATM resides at sites in the cytoplasm as well as the nucleus and co-localizes with catalase in peroxisomes; increased lipid peroxidation and decreased catalase has been detected in AT-deficient cells. Markers of oxidative stress are increased in both AT heterozygotes and homozygotes. Activation of JNK and the AP-1 pathway is present in the brains of ATM-deficient mice. Self-renewal of hematopoietic stem cells requires the inhibition of reactive oxygen species generation by ATM .
p53 and Atherosclerosis
How ATM modulates responses to oxidative stress is unknown but it is reasonable to assume that p53 is involved. The tumor suppressor p53 responds to DNA damage by inducing an increasingly complex series of events, including apoptosis and cell cycle arrest (which appear to be transcription-dependent), as well as control of DNA repair and recombination (which may be transcription-independent). In response to stress, activated ATM phosphorylates p53 and MDM2, which leads to an increase in p53 protein levels and activity. While a role for ATM in atherosclerosis has not been firmly established, several studies suggest that p53 may be involved in vascular disease. p53 (and MDM2) are present in human atherosclerotic lesions. In rabbits, diet-induced atherosclerosis is associated with oxidation-induced DNA damage and the induction of p53 in the vasculature. In the apoE null model, p53−/− mice have increased atherosclerosis associated with accelerated cell proliferation without an effect on apoptosis. The p53 effect appears to be mediated in part by macrophages, since the transplantation of p53 null bone marrow in both apoE*3-Leiden mice (an animal model for human-like atherosclerosis described by van Vlijmen et al., Circ Res., 2001, 88(8):780-6) as well as LDL receptor null mice results in more atherosclerosis. Recent evidence suggests that the anti-atherosclerotic effect of p53 in dietary models may be complex, promoting apoptosis in macrophages and preventing apoptosis in smooth muscle cells. The protective effect may be limited to diet-induced atherosclerosis models. Using a plaque-rupture model involving phenylephrine administration, one group reported that adenoviral-mediated overexpression of p53 in smooth muscle cells increased apoptosis and destabilized lesions. Another growth suppressor, p27 (a cyclin-dependent kinase inhibitor), has been shown to decrease diet-induced atherosclerosis by decreasing macrophage proliferation, but p21 (a different member of the same cyclin-dependent kinase inhibitor family) increases atherosclerosis. In short, recent studies have shown that ATM is an important activator of p53, and p53 is likely to have anti-atherogenic effects in the vasculature.
ATM and the Antimalarial Drug Chloroquine
Inactive ATM exists as a dimer in cells. In response to stress, ATM phosphorylates itself, a modification that does not affect the intrinsic kinase activity of the molecule, but instead dissociates the dimer and allows substrates access to the kinase domain of the molecule. This phosphorylation, representing ATM activation, occurs at serine 1981 of ATM and is sensitive to cellular stress. Low dose irradiation producing as few as four strand breaks in the entire genome and experiments using manipulated mammalian cells following induction of only two well-defined DNA strand breaks have been shown to cause ATM phosphorylation. Thus, it is believed that ATM activation does not require physical contact with DNA. Instead, since strand breaks alter chromatin structure, ATM is probably capable of sensing subtle changes in chromatin structure. Chromatin structure can be altered without inducing DNA strand breaks by several manipulations including exposure to mildly hypotonic media, inhibitors of histone deacetylase, and exposure to the antimalarial drug chloroquine.
Chloroquine
Chloroquine is a DNA intercalating agent that functions as a mild topoisomerase II inhibitor. Chloroquine is used to prevent and treat malaria, a red blood cell infection with plasmodium species of protozoa transmitted by the bite of a mosquito, and to treat parasitic conditions such as liver disease caused by other protozoa (tiny one-celled animals).
Chloroquine and related aminoquinolines have also found use in the treatment of other chronic inflammatory diseases. Antimalarials were first described as treatments for rheumatologic disease in 1894, and the class gained acceptance for use in inflammatory conditions following a 1951 report in the Lancet. Chloroquine and related aminoquinolines have been shown to be effective for treating systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). These anti-inflammatory effects have led to their use as prophylaxis for deep venous thrombosis and treatment of sarcoidosis.
Chloroquine and Metabolism
Effects of chloroquine on insulin sensitivity were reported in 1984, when high dose chloroquine (1000 mg/day) was used in a patient to successfully reverse severe insulin resistance thought to be due to accelerated degradation of insulin. Subsequent studies confirmed a modest effect of glucose lowering that was proportional to the degree of insulin resistance, i.e., there was essentially no effect in non-diabetic subjects and the greatest effect was seen in those who were most insulin resistant, when administering high dose chloroquine. Chloroquine has been reported to increase the affinity of the insulin receptor and increase insulin secretion by isolated islets, both of which may reflect a global increase in insulin signaling. There is also a report of a hyperinsulinemic-euglycemic clamp study in patients with type 2 diabetes that demonstrated that administering high dose chloroquine (1000 mg/day) for three days modestly decreased insulin resistance in peripheral tissues without affecting endogenous glucose production (Powrie et al., Am. J. Physiol., 1991, 260:897-904). Rahman et al. (J. Rheumatol., 1999, 26(2):325-30), reported that antimalarials lower total cholesterol in patients also receiving steroids and may minimize steroid induced hypercholesterolemia in patients with systemic lupus erythematosus. Munro et al. (Ann. Rheum. Dis., 1997, 56:374-377) reported that for a test group of 100 rheumatoid arthritis patients, the group treated with oral hydroxychloroquine had a significant overall improvement in their lipid profile. Wallace (Lupus, 1996, 5 Suppl 1:S59-64) concluded that chloroquines are safe and effective as a therapy for selected patients having any one of the following disorders: porphyria cutanea tarda, cutaneous sarcoidosis, cutaneous manifestations of dermatomyositis, hyperlipidemias, and thromboembolic prophylaxis for patients with antiphospholipid antibodies.
Insulin resistance is widely held to explain the association between the metabolic syndrome and vascular disease. However, insulin resistance may not directly cause atherosclerosis (Semenkovich, 2006, J. Clin. Invest. 116:1813-1822). Insulin resistance is not related to vascular lesions after correcting for glucose tolerance. Pioglitazone, an insulin sensitizer, does not decrease cardiovascular events in patients with insulin resistance. Studies directly addressing the role of macrophage insulin resistance in atherosclerosis are conflicting. As opposed to representing a unique entity, the metabolic syndrome may simply reflect the cumulative contribution of its components to atherosclerotic risk.
Historically, chloroquine has been used with caution because it can cause retinal toxicity. There are numerous reports addressing chloroquine toxicity that can be summarized as follows: reports that retinopathy is rare and the drug can be taken for years without toxicity; hydroxychloroquine, a less effective anti-inflammatory agent, can also cause retinopathy, although the risk is lower than with chloroquine; retinopathy risk is dose-dependent with the lowest cumulative dose associated with retinopathy being 125 grams (several groups have shown no effects despite cumulative doses of 300 grams); and doses of about 250 mg per day or 3.5 mg/kg per day are considered safe for chronic treatment.
Presently there is no one treatment for the combination of symptoms that make up metabolic syndrome. Thus, there is a need for an effective, safe treatment for the combination of disorders associated with metabolic syndrome. The present invention provides novel methods and compositions comprising low doses of a chloroquine compound to modulate ATM activity and to alleviate or prevent the numerous symptoms of metabolic syndrome. Compounds and methods of the present invention may also be utilized in combination with one or more other treatments such as antihyperglycemic diabetes treatment, an antihypertensive agent, an antithrombotic agent, and/or an inhibitor of cholesterol synthesis or absorption to augment these treatments.